CN111607605A

CN111607605A - Construction method of multivalent epitope and subunit vaccine

Info

Publication number: CN111607605A
Application number: CN202010482108.4A
Authority: CN
Inventors: 马永平
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2020-05-27
Filing date: 2020-05-29
Publication date: 2020-09-01
Anticipated expiration: 2040-05-29
Also published as: CN111607605B

Abstract

The invention discloses a construction method of a multivalent epitope and subunit vaccine, belonging to the field of vaccines. According to the invention, the antigen protein is connected to two ends of LTB26 through fusion expression to obtain the fusion protein, and then the pentamer of the fusion protein is obtained by virtue of the characteristic that LTB26 can be self-assembled to form the pentamer. The active ingredient of the vaccine of the present invention is just a pentamer of the aforementioned fusion protein. The vaccine of the invention has abundant quantity and variety of immunogens, and combines the activity of the LTB26 immunoadjuvant with the immunogen activity of the antigen peptide, so that the vaccine can stimulate an organism to generate a large amount of specific antibodies, excite effective immunoreaction, and save the procedure of additionally adding the immunoadjuvant. Fusion of LTB26 to both ends can be other proteins besides antigen protein, and can be applied to fields other than vaccine preparation, such as mass preparation of antibodies, development of medical detection kits, and the like.

Description

Construction method of multivalent epitope and subunit vaccine

Technical Field

The present invention is in the field of vaccines.

Background

Immunization (immunization) vaccination is to inoculate a vaccine in a healthy human body so that a vaccinee can generate antibodies aiming at specific pathogens under the condition of no disease attack, and specific immunity to specific diseases is obtained, thereby achieving the purpose of preventing and treating certain infectious diseases. According to different inoculation sites, the method is divided into invasive immunization (such as injection, scratch and the like) and noninvasive immunization (such as nasal drip, spray and the like).

At present, the development of vaccines mainly comprises 5 technical routes of inactivated vaccines, attenuated live vaccines, recombinant genetic engineering vaccines, viral vector vaccines and nucleic acid vaccines [ jiabingice, the plum country, history and prospect of vaccine technical development [ J ]. biological report, 2016, 51 (06): 1-3. The traditional inactivated vaccine and attenuated live vaccine are developed for a long period, and the requirement of preventing and treating the emergent infectious diseases is difficult to quickly respond. The inactivated vaccine cannot generate cellular immunity and mucosal immunity, the protection on pathogens infected by mucosa is not high, the antibody titer of the antigen of the inactivated vaccine is reduced along with time, multiple times of reinforced inoculation are needed, the required antigen amount is large, and the cost is high. Protein subunit vaccines containing only antigen and more optimized and reduced fourth generation epitope (epitope) vaccines can overcome some of the disadvantages of the above vaccines [ giardia ice, lie country. history and prospect of vaccine technology development [ J ] biological bulletin, 2016, 51 (06): 1-3 ], however, protein subunit vaccines are less immunogenic and require more excellent immunoadjuvants.

The lack of efficient and safe human adjuvants is the biggest problem in current vaccine research, at present, over 98% of human vaccine adjuvants are aluminum salt adjuvants (aluminum adjuvants for short), and the greatest defect of vaccines containing aluminum adjuvants is that the human vaccine adjuvants can not be freeze-dried or stored in a freezing environment at 2-8 ℃, and once the human vaccine which is not stored at 2-8 ℃ and transported in a cold chain is inoculated, the first risk is that the human vaccine is ineffective in immunity. But also can not induce Th1 type cellular immune response, can not activate CTL to eliminate tumor cells and intracellular pathogen infected cells; has the side effect of inducing Ig E mediated type I hypersensitivity; the general reactions such as fever, diarrhea, dizziness, vomiting, etc. may also occur in the case of sore pain at the site of inoculation [ jiabingice, li wei. 1-3. Another 3 FDA approved personal adjuvants are MF59 (a mixture of 1% squalene, 0.5% Tween 80 and 0.5% polysorbate trioleate) and AS03 (a mixture of vitamin E, Tween 80 and squalene) and AS04(monophosphoryl lipid a (MPLA) formulated with aluminium salts)), which improve the disadvantages of aluminium adjuvants, but have a limited range of use.

In analyzing SRAS from 2003, avian influenza infection in 2005, H5N1, hand-foot-and-mouth disease in 2008, H1N1 influenza a in 2009, MERS in 2012, H7N9 avian influenza pandemic in 2013, and new crown pneumonia pandemic to the end of 2019, we found three laws: firstly, people are infected by epidemic infectious diseases of strong pathogenic animals continuously; illustrating that human fight against infectious diseases caused by known and unknown viruses will be long-term. Secondly, viral infectious diseases are the main factors, and the embarrassment that no specific medicine (including vaccine) can treat is faced. For example, the death rate of H5N1 avian influenza in 2005 is 71.4%, and the death rate in 2006 is 66.7%. Thirdly, according to the analysis of infection sites and infection routes, most of viral virulent infectious diseases which are epidemic in recent years are affected by the respiratory mucosa system infected by droplets (the hand-foot-and-mouth disease virus can also be transmitted through the digestive tract mucosa). In The example of COVID-19, in addition to infection of respiratory and pulmonary tissues, intestinal mucosal tissues are also infected with diarrheal symptoms [ Shang W, et., The outbreak of SARS-CoV-2 pneumoconia caps for viral vaccines. NPJVACCines.2020Mar 6; 5: 18. doi: 10.1038/s 41541-020-. The mucosal immune system is the first line of defense against pathogenic microorganisms at mucosal sites, and is also the main target for designing mucosal immune vaccines. Therefore, we believe that induction of mucosal immunity through development of mucosal immunity vaccines is the best approach to prevent such infectious diseases. In view of this, the development of a general platform for mucosal immunization vaccines against highly pathogenic pathogens (e.g., viruses) has a very broad application prospect. The solution of mucosal immunoadjuvant is the foundation of the foundation.

LT protein belongs to A-B type bacterial protein toxin family, and is composed of a toxic A subunit (LTA) and five B subunits (LTB) which are aggregated into a ring, wherein the LTB can be combined with cell surface GM1 and TLR2 receptor, and has mucosal immune adjuvant activity. LTB26 is a mutant LTB, and it has been reported that LTB26 combined with VP8 can greatly improve the immunological response intensity of the body to VP8 (study on mutant adjuvant activity and action mechanism of Escherichia coli heat-labile enterotoxin B subunit (LTB) [ D ].2017 ].

Disclosure of Invention

The invention aims to solve the problems that: provides a construction method of a multivalent epitope and subunit vaccine based on LTB 26.

The technical scheme of the invention is as follows:

a method of constructing a multivalent epitope and subunit vaccine, the method comprising:

fusing the gene coding the antigen epitope or subunit at both 3 'and 5' ends of the gene coding LTB26 to obtain a fused gene, and fusing the fusion protein coding the antigen epitope or subunit at both ends of the expressed LTB26N and C.

A fused gene obtained by fusing a gene encoding an epitope or subunit to both 3 'and 5' ends of a gene encoding LTB 26;

preferably, the gene encoding the epitope or subunit is 24 to 1500nt in length.

The amino acid sequence of the epitope is shown in SEQ ID NO.5 and 6.

The base sequence of the gene for coding the epitope is shown in SEQ ID NO.3 and 4.

A recombinant plasmid, wherein the recombinant plasmid is obtained by constructing the fusion gene on a gene expression vector.

The recombinant plasmid is obtained by constructing the fusion gene on pET32a vector.

A fusion protein obtained by expressing the fusion gene.

A protein pentamer which is a pentamer formed by self-assembly of the aforementioned fusion protein.

Use of the fusion gene, the plasmid, the fusion protein or the protein pentamer for the preparation of an antibody or a vaccine.

An LTB 26-based subunit vaccine, characterized by: the vaccine comprises the fusion protein and/or a pentamer of the fusion protein as an active ingredient.

In the present invention, "protein" is a protein in a broad sense, and refers to a dehydration condensate of amino acids of any length, and conventional "oligopeptide" and "polypeptide" are also within the definition of "protein" in the present invention.

The invention has the beneficial effects that:

the fusion protein obtained by fusing the gene of the encoding antigen protein on the upstream and downstream of the gene of LTB26 can be automatically assembled into a pentamer, thereby amplifying the number of antigen molecules and directly enhancing the antigen dosage.

The vaccine of the invention has large immunogen quantity (each pentamer can have 10 same antigen peptides at most) and abundant varieties (each pentamer can have 10 different antigen peptides at most), and combines the activity of LTB26 immune adjuvant and the immunogen activity of antigen peptide/antigen epitope (such as the antigen epitope of novel coronavirus), so that the vaccine can stimulate an organism to generate a large amount of specific antibodies and stimulate effective immune response.

In addition, in the field of antibody production, fusion of monovalent antigens at two ends of LTB26 can be used for stimulating immune cells to produce more antibodies, and the effect is obviously better than that of the antigen used alone. These antibodies can be used for protein isolation or detection in the field of scientific research or medicine.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: LTB 26-recombinant subunit bivalent vaccine format.

FIG. 2: analysis of the immune efficacy (specific IgG titres) of EP1-LTB26-EP 2.

FIG. 3: LTB 26-recombinant subunit pentavalent vaccine format.

FIG. 4: LTB 26-recombinant subunit ten vaccine format.

Detailed Description

Experimental materials and reagents: pET32a-LTB26-6 XHis, and pGEX-LTB26 plasmids were constructed and stored by the university of Chongqing medical Biochemical and molecular biology research laboratory; prokaryotic expression vectors pET32a, pGEX, e.colitop10, e.colibl21(DE3) were stored by the university of chongqing medical science biochemistry and molecular biology research laboratory; male rats were provided by the experimental animals center of the university of chongqing medicine. T4 ligase, Taq common enzyme, SalI, SacI, BamHI, Not I, protein Marker, DNAmarker, Plasmid Mini Kit I, Cycle-Pure Kit, gel recovery Kit, IPTG, ampicillin, BCA protein concentration determination Kit, PMSF and other reagents.

Example 1 preparation of EP1-LTB26-EP2 fusion protein (bivalent vaccine):

1. recombinant expression

1.1 cloning and expression of pET32-RBD EP1-LTB26-RBD EP2 Gene

a. Synthesizing a recombinant target gene:

inserting LTB26 gene (SEQ ID NO.2) between SacI (GAGCTC) of DNA sequence (SEQ ID NO.1) of Receptor Binding Domain (RBD) of novel coronavirus (SARS CoV-2) and SalI (GTCGAC) enzyme cutting site, and connecting epitope EP1(SEQ ID NO.3) and EP2(SEQ ID NO.4) of novel coronavirus at two ends of LTB26 to form EP1-LTB26-EP2 fusion gene. The EP1-LTB26-EP2 fusion gene is constructed on a common Escherichia coli expression vector (such as pET32 a).

The related sequence is as follows:

DNA sequence encoding RBD (SEQ ID NO. 1):

in SEQ ID NO.1, the underlined portions indicate the cleavage sites SacI (GAGCTC) and SalI (GTCGAC).

LTB26 gene (SEQ ID NO. 2):

the DNA sequence (SEQ ID NO.3, length 100bp) encoding EP1 is specifically:

the DNA sequence (SEQ ID NO.4, length 117bp) encoding EP2 is specifically:

the amino acid sequence of EP1(SEQ ID NO.5) is: FGEVFNATRFASVYAWNRKRI are provided.

The amino acid sequence of EP2(SEQ ID NO.6) is: SNCVADYSV LYNSASFSTFKCYGVS are provided.

b. Transforming Escherichia coli to prepare recombinant bacteria

(1) Mixing 20ul of the above pGEX-LTB26, pET32-EP1-LTB26-EP2 and plasmid DNA with 50. mu.l of E.coli.BL21 competent cells, respectively, and standing on ice for 30 minutes;

(2) heat shock at 42 ℃ for 90 seconds, ice bath immediately for 1 minute;

(3) 50 μ l of the above-mentioned transformed bacterial liquid was mixed well, applied to LA (LB containing 100ug/mL Amp) plates, and cultured in an incubator at 37 ℃ overnight in an inverted manner.

(4) Colonies of moderate size were selected on LA plates and positive clones were initially screened by colony PCR.

(5) Correct plasmid-company sequencing was identified.

1.2 inducible expression of recombinant E.coli

(1) Inoculating the recombinant bacteria to LA culture medium (100 mug/ml Amp), and shake culturing at 37 deg.C for 12h at 250 r/min;

(2) transferring the bacterial liquid to fresh LA culture medium at an inoculum size of 1: 100, continuously culturing in a shaker at 37 deg.C at a rotation speed of 200r/min, detecting in real time with spectrophotometer, and detecting when bacterial liquid OD₆₀₀When the value reaches 0.6-0.8 (bacterial logarithmic growth phase), IPTG is added to make the final concentration 0.5mmol/L, and the induction expression is carried out for 5h at 37 ℃.

(3) The induced expression bacteria liquid is centrifuged for 10min at 13000g, the supernatant is discarded, the bacteria is fully resuspended by the bacteria lysate, and the lysis treatment is carried out for 30min at room temperature.

(4) The supernatant and the precipitate were collected by centrifugation, and the lysed specimen was analyzed by 12% SDS-PAGE.

1.3 separation and purification

The recombinant protein expression bacteria are cracked by a reagent in a magnetic bead purification kit (Suzhou beaver nanometer science and technology company), centrifuged for 10min at 2000rpm, the supernatant is respectively mixed with 1ml of magnetic beads, acted for 30min at room temperature, placed in a magnetic separator for separation, the supernatant is discarded, the magnetic beads are washed by washing liquid for 10min, the supernatant is discarded, the magnetic beads are washed by eluent for 5min, centrifuged for 10min at 2000rpm, and the supernatant is taken, namely the pure product EP1-LTB26-EP2 fusion protein and stored for later use at-20 ℃.

Typically, LTB26 self-assembles into pentamers during expression in e.coli; in this example, one LTB26 pentamer carried 5 identical EP1 and EP2 epitopes, increasing the number of single antigenic peptides by 5-fold and enhancing the immunogenicity of the epitope (fig. 1).

Experimental example 2 safety test of the bivalent vaccine polypeptide EP1-LTB26-EP2 of the present invention

1. Experimental methods

The safety of the rat was tested by injecting EP1-LTB26-EP2 prepared in example 1 of the present invention into the abdominal cavity of the rat using male rats as animal models:

a) 6 healthy male rats with a body weight of 3-4 weeks old were collected from the experimental animal center of Chongqing medical university.

b) EP1-LTB26-EP2 having immunoadjuvant activity prepared in example 1 was dissolved in PBS buffer.

c) 0.50ml of EP1-LTB26-EP2 (without exogenous endotoxin detection and treatment) with the concentration of 1.0 mu g/ml, 2.0 mu g/ml, 5.0 mu g/ml, 10.0 mu g/ml, 20.0 mu g/ml, 50.0 mu g/ml and 100.0 mu g/ml are intraperitoneally injected into the rats in the a), the difference of signs (such as activity, feeding capacity, tremor, piloerection, fecal diarrhea and the like) between experimental rabbits and controls is observed within 3 days, and the survival condition is observed within 3 days, and 3 injections are performed in total within 3 days.

d) Animals after the 3 rd injection were sacrificed by anesthesia 8 hours later, and the heart, liver, spleen and kidney were taken for pathological examination, while the above tissues of normal rabbits were used as controls.

2. Results of the experiment

(1) No change of the mobility and the feeding ability of experimental rats is found within 3 hours after injection, no difference and change of other physical signs such as tremor, piloerection and feces diarrhea are caused, and no dead individual is caused.

(2) After entering the animal body, EP1-LTB26-EP2 has no pathological damage to the major organs such as heart.

The experimental results show that EP1-LTB26-EP2 of the present invention is safe for in vivo use.

Example 3 testing of the effectiveness of the bivalent vaccine EP1-LTB26-EP2 according to the invention

1. Experimental methods

The EP1-LTB26-EP2 fusion protein having immunoadjuvant activity prepared in example 1 was dissolved in PBS buffer. The effectiveness detection is carried out according to the following steps:

a) rats were purchased at the animal testing center at Chongqing university of medicine (male) at 18, randomly divided into 4 groups of 6 rats each.

b) EP1-LTB26-EP2 fusion protein prepared in example 1 (6.0. mu.g/mouse) was subjected to nasal drop immunization on 4% chloral hydrate anesthetized rats (1.0ml/100 g); PBS was applied to the nose as a control.

c) B) performing a second booster immunization at the same dose as b) on 7 th day after the primary immunization, and performing a third booster immunization on 14 th day, wherein a blood sample is collected before each booster immunization, and is stored at-80 ℃ for later use. Blood samples after the third booster immunization were collected on day 21 and all experimental animals were sacrificed under anesthesia and stored at-80 ℃ for future use.

d) The specific IgG antibodies against EP1 and EP2 in blood samples were detected by ELISA.

2. Results of the experiment

High levels of specific antibodies were detected in EP1 and EP2 in animals mixed with the EP1-LTB26-EP2 fusion protein of the present invention, compared to the control group (FIG. 2). In addition, immunization of animals with EP1 and EP2 alone as antigens failed to produce specific IgG antibodies.

The experimental results show that a recombinant subunit bivalent vaccine can be constructed by fusing two different epitopes of EP1 and EP2 to the N-terminal and C-terminal of LTB26 at the same time. The fusion protein obtained by connecting the antigenic peptide or the antigenic epitope to the two ends of LTB26 can form a pentamer to generate a high-level specific antibody, and the feasibility and the high efficiency of the method are proved.

Example 4 Pn-LTB26-Pn recombinant subunit pentavalent vaccine construction model

Genes encoding antigenic peptides P1, P2, P3, P4 or P5 are respectively connected to the upstream and downstream of 5 LTB26 gene fragments (each LTB26 fragment is connected with one antigenic peptide) to obtain five fusion fragments, the fusion fragments are respectively connected with signals of a promoter and a terminator, and then are connected in series between Sac I and Sal I sites of a pET32 vector to obtain an expression vector pET32-Pn-LTB26-Pn (n is 1, 2, 3, 4, 5), and after expression and purification, a recombinant subunit pentavalent vaccine model formed by self-assembly of recombinant proteins is obtained (figure 3).

Example 5 Pn-LTB-Px recombinant subunit decavalent vaccine model

Genes encoding antigenic peptides P1, P2, P3, P4 or P5 were ligated to the upstream and genes encoding antigenic peptides P6, P7, P8, P9 and P10 to the downstream of 5 LTB26 gene fragments, respectively, to obtain five fusion fragments, and after ligation of promoter and terminator signals, the fusion fragments were constructed in tandem between Sac I and Sal I sites of the pET32 vector, to obtain pET32-Pn-LTB26-Px (n ═ 1, 2, 3, 4, 5; x ═ 6, 7, 8, 9, 10) expression vectors, and a recombinant subunit ten-valent vaccine model (fig. 4) in which recombinant proteins were self-assembled was obtained.

The essence of the invention is still not changed by changing the tandem fusion expression shown in examples 3-5 into the fusion expression of each antigen with LTB26 alone, and then mixing the fusion expression and performing denaturation-renaturation to assemble bivalent or multivalent vaccine respectively, and the invention belongs to the conventional alternative form of the invention.

By replacing the LTB26 subunit of the invention with a subunit of the B5 pentamer of the AB5 pattern of other bacterial and plant toxins, the essence of the invention remains unchanged and is a routine alternative to the present invention.

In conclusion, the invention adopts the principle that LTB26 can form pentamer, and antigen peptide is fused at two ends of LTB26 to form monovalent or multivalent (two, three, four, five, six, seven, eight, nine or ten valent) subunit vaccine. The vaccine is rich in quantity and variety of immunogens, and combines the activity of the LTB26 immunoadjuvant with the immunogen activity of the antigen peptide, so that the vaccine can stimulate an organism to generate a large amount of specific antibodies to stimulate effective immunoreaction. In the field of antibody production, fusion of monovalent antigens at two ends of LTB26 can be used for stimulating immune cells to produce more antibodies, and the effect is obviously better than that of the antigen used alone.

SEQUENCE LISTING

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Claims

1. A method for constructing a multivalent epitope and subunit vaccine is characterized in that: the method comprises the following steps:

the gene coding the antigen epitope or subunit is fused at the 3 'end and the 5' end of the gene coding LTB26 to obtain a fusion gene, and the fusion protein coding the antigen epitope or subunit is fused at the two ends of the expressed LTB26N, C.

2. A fusion gene, characterized by: it is a fusion gene obtained by fusing genes encoding epitope or subunit at both 3 'and 5' ends of the gene encoding LTB 26;

preferably, the gene encoding the epitope or subunit is 24 to 1500nt in length.

3. The fusion gene of claim 2, wherein: the amino acid sequences of the antigen epitopes are shown as SEQ ID NO.5 and 6.

4. The fusion gene of claim 3, wherein: the base sequence of the gene for coding the epitope is shown in SEQ ID NO.3 and 4.

5. A recombinant plasmid, characterized in that: the recombinant plasmid is obtained by constructing the fusion gene of any one of claims 2 to 4 on a gene expression vector.

6. The recombinant plasmid of claim 5, wherein: the plasmid is obtained by constructing the fusion gene of any one of claims 2 to 4 on a pET32a vector.

7. A fusion protein, characterized in that: it is a protein expressed from the fusion gene according to any one of claims 2 to 4.

8. A protein pentamer characterized by: it is a pentamer formed by the self-assembly of the fusion protein polypeptide of claim 7.

9. Use of the fusion gene according to claims 2 to 4, the plasmid according to claim 5 or 6, the fusion protein according to claim 7 or the protein pentamer according to claim 8 for the preparation of an antibody or a vaccine.

10. A multivalent epitope and subunit vaccine, characterized by: the vaccine contains the fusion protein of claim 7 and/or the pentamer of the fusion protein of claim 8 as an active ingredient.